1. Field of the Invention
The invention relates to improved methods for secretion of proteins through the bacterial membrane including the use of filamentous phage coat proteins for display or exogenous proteins including libraries of variants which may be monomeric, dimeric, or heterodimeric or multimeric proteins including complete antibodies and antibody fragments, or other disulfide linked multimeric constructs by coupling a mutant pelB or mutant ompA secretion signal to the exoprotein construct.
2. Discussion of the Field
Filamentous phage display is a widely used technology for affinity-based selection of proteins as each phage particle links the nucleic acid encoding the polypeptide fused to the N-terminus of its coat protein together in the selection process. M13 bacteriophage encodes five coat proteins with approximately five copies of the minor coat proteins pIII and pVI at one end of the phage and the same number of pVII and pIX at other end of the phage. The phage DNA is encapsulated by approximately 3000 copies of the major coat protein, pVIII. Although the display of foreign polypeptides has been accomplished with each of the coat proteins of M13, pIII and pVIII are by far the most common fusion partners. Using this technique, libraries of peptides, Fabs, scFvs and other protein binders have been constructed and found use in diverse applications and with great commercial value.
The pIII coat protein has been favored over the other proteins is due to its length and conformation. The pIII minor coat protein is a 402 amino acid, 42 kD protein responsible for phage infection into E. coli comprising two domains connected by a flexible hinge. Fusions to the pIII N-terminus tether the displayed protein away from the phage particle reducing its ability to interfere with required pIII functions and further providing adequate access for ligand binding. In contrast, pVII and pIX are short helical proteins of 33 and 32 aa, respectively, closely packed in the phage particle. Nevertheless, there have been reports describing the display of scFv libraries on pIX (Gao, C. et al. Proc Natl Acad Sci USA 99, 12612-12616, 2002) and heterodimeric display of Fv or Fab libraries by taking advantage of the ability to fuse different polypeptides to both pVII and the closely adjacent pIX (Gao, et al. 1999 Proc Nat Acad Sci 96: 6025-6030 and Janda U.S. Oat. No. 7,078,166). Phage displaying pVII fusions have also been reported (Kwasnikowski, et al. 2005. J Immunol Methods 307:135). Using a hybrid phage vector or phagemid vectors peptides, Fab, and other proteins can be displayed on phage fused to the pIX coast protein (WO2009/085462, WO2009/085464, and WO2009/085468). An alternative approach in which exoproteins encoded by the phage or phagemid vector are not fused to the coat protein but rather covalently attach to re-engineered coat proteins pIII and pIX with through disulfide bonding has also been described (U.S. Pat. No. 6,753,136).
Escherichia coli is one of the most widely used hosts for the production of recombinant proteins. However, there are often problems in recovering substantial yields of correctly folded proteins. One approach to solve these problems is to have recombinant proteins secreted into the periplasmic space or culture medium. The secretory production of recombinant proteins has several advantages, such as simplicity of purification, avoidance of protease attack and N-terminal Met extension, and a better chance of correct protein folding.
After DNA replication and expression of phage proteins, M13 and other filamentous phage are assembled at the bacterial host cell membrane. The transport of the phage structural proteins into the membrane is a crucial step for phage assembly. In the practice of phage display, bacterial signal peptides have been widely used in constructs to help transport fusion proteins through the membrane and, have been critical to achieving display of certain of types of fusion proteins. The signal peptide of pectate lyase B (pelB) from Erwinia carotovora has been widely utilized in for the purpose of both enhancing protein production using bacterial cells as well as in phage display.
The ability to display a more diverse and complex proteins such as dimeric proteins by bacterial culture or on the surface of a phage particle using bacterial host cells and in a combinatorial library format is advantageous in being able to perform selections of modified and re-engineered complex protein structures. There is a continuing need to advance the art for generating highly efficient protein production methods as well as high throughput methods of screening variants of complex proteins such as that of the human IgG, which is a homodimer of heavy and light chain pairs (heterodimers) connected via intermolecular disulfide bonds.